The long-term goal of this research is to elucidate the molecular and cellular mechanisms underlying the effects of ethanol on the cyclic adenosine monophosphate (cAMP) signaling pathway in the central nervous system. cAMP signal transduction has been postulated to play a critical role in the physiological and behavioral responses to ethanol in animals and in the development of and predisposition to alcoholism in humans. The cAMP signaling system can be modulated by both acute and chronic ethanol exposure. The effect of ethanol on the levels of cAMP was measured by immunological and radiochemical methods in past studies, which had poor temporal resolution and no spatial resolution. Recently, single polypeptide chain cAMP sensor molecules have been developed, which can monitor cAMP levels by fluorescence resonance energy transfer (FRET). Using these sensor molecules it is possible to study real-time dynamic changes in the concentration of cAMP at a subcellular level with high spacio-temporal resolution. Emerging experimental evidences support the concept that subcellular compartmentalization is critical for cAMP signaling. We will utilize this technology for studying the effects of ethanol on the cAMP signaling system by testing the hypothesis that ethanol influences cAMP metabolism in a subcellular compartment specific manner. In Specific Aim 1, we will determine the effects of ethanol on cAMP metabolism in HeLa cells. Using a FRET-based cAMP sensor, Epac1-camps, the level of cAMP in HeLa cells will be monitored in real-time at the single cell level in the absence and presence of ethanol. Key observations of ethanol effects on cAMP metabolism studied by radiochemical methods in the past will be confirmed. Epac1-camps will be genetically modified so that the sensor can be targeted to the nucleus, plasma membrane, and cytoplasm. The effects of ethanol on cAMP in these three compartments will be examined. In Specific Aim 2, we will determine the contributions of phosphodiesterase, protein kinase A, and A-kinase anchoring proteins on the observed effects of ethanol. In the presence of pharmacological reagents specific to these proteins, ethanol's effect on cAMP metabolism in the three compartments will be examined by the FRET sensors. In Specific Aim 3, we will determine the effects of ethanol on cAMP metabolism in subcellular compartments of neuronal cells in primary culture. Primary rat neurons isolated from the cerebral cortex and striatum will be transfected with Epac1- camps targeted to different subcellular compartments (nucleus, plasma membrane, and cytoplasm). The levels of cAMP will be monitored in high spacio-temporal resolutions in the absence and presence of ethanol. The knowledge and technical expertise we will gain during the course of this study is indispensable for future studies of ethanol's effects on cAMP metabolism in the brain. Project Narrative Cyclic adenosine monophosphate (cAMP) signal transduction has been postulated to be a contributing factor to the development of and predisposition to alcoholism in humans. The knowledge and technical expertise we will gain during the course of this study is indispensable for understanding the effects of alcohol on cAMP metabolism in the brain, and may lead to the development of a new generation of tissue- and cell- specific drugs.